The present invention relates to methods of treating a subject suffering from infection with Mycobacteria by administration of Bactericidal/Permeability-Increasing Protein (BPI) protein products. Mycobacterium is a non-motile, acid-fast, aerobic, genus of bacteria known to cause grave human and animal diseases, such as tuberculosis and leprosy. Infections caused by M. avium are the most common form of disseminated bacterial disease in AIDS patients. Orme, et al., Infect. and Immun., 61(1):338-342 (1993).
The administration of conventional antibiotics to treat Mycobacterial infection is known in the art and has achieved varying success depending on the susceptibility of the bacterial strain, the efficacy and toxicity of the antibiotic(s) employed, the duration of treatment, and numerous other factors. Antimicrobials that have been employed alone or in combination to treat Mycobacterial infections, including those caused by M. tuberculosis include isoniazid, rifampin, ethambutol, p-aminosalicylic acid, pyrazinamide, streptomycin, capreomycin, cycloserine, ethionamide, kanamycin, amikacin, amithiozone, rifabutin, clofazimine, arithromycin, clarithromycin, ciprofloxacin and ofloxacin. McClatchy, Antimycobacterial Drugs: Mechanisms of Action, Drug Resistance, Susceptibility Testing, and Assays of Activity in Biological Fluids, pp. 134-197, In Antibiotics in Laboratory Medicine, 3rd ed., V. Lorian, ed. The Williams and Wilkins Co., Baltimore (1991). As many Mycobacterial strains are drug resistant, serious obstacles exist for control and successful treatment of tuberculosis and other Mycobacterial diseases. Id.
A variety of factors have made treatment of individuals afflicted with Mycobacterial diseases problematic. First, Mycobacteria possess a very hydrophobic cell wall that affords protection against the host""s immune system. As Mycobacterial infections tend to be chronic, the pathologies of these organisms are generally due to host response. Also, many Mycobacterial strains are drug-resistant. These and other factors make the development of novel, effective methods for treating Mycobacterial diseases highly desirable.
Mycobacteria are readily distinguished from gram-negative and gram-positive bacteria by acid fast staining due to significant differences in cell wall structure. Gram-negative bacteria are characterized by a cell wall composed of a thin layer of peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide (LPS), whereas gram-positive bacteria have a cell wall with a thicker layer of peptidoglycan with attached teichoic acids, but no LPS. The Mycobacterial cell wall is rich in fatty acids, including a major constituent, lipoarabinomannan (LAM), which is widely distributed within the cell wall of Mycobacterium species. LAM has been purified from both M. leprae and M. tuberculosis. Hunter et al., J Biol. Chem., 261:12345-12351 (1986). LAM is a serologically active mannose containing phosphorylated lipopolysaccharide that may be membrane associated.
The complex physiological effects of LAM appear to be concentration, time, and source-dependent. For example, Chaterjee et al., Infect. and Immun., 60(3):1249-1253 (1992), reported that, in the first 24 hours following exposure, LAM from an avirulent strain of tuberculosis was 100-fold more potent at stimulating TNF secretion in mouse macrophages than LAM from a virulent strain. LAM concentrations of 0.01-10 xcexcg/ml for the avirulent strain and 0.01-100 xcexcg/ml for the virulent strain were tested, and increased LAM concentration was associated with increased TNF production with LAM from both species.
Macrophage-inhibitory effects of LAM have also been described in the art. LAM purified from both M. leprae and M. tuberculosis has been reported to be a potent in vitro inhibitor of T-cell lymphokine activation of mouse macrophages. Sibley et al., Infection and Immunity, 56(5):1232-1236 (1988). Because the principle efferent role of the macrophage in acquired resistance to intracellular pathogens requires activation by T-cell lymphokines, notably gamma-interferon (IFN-xcex3), macrophages whose activation-response is inhibited are severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities.
In another study, Sibley et al., Clin. Exp. Immunol., 80(1):141-148 (1990), reported that pretreatment of mouse macrophages with 50 to 100 ug/ml LAM blocked macrophage activation by IFN-xcex3, but pretreatment with 10 xcexcg/ml LAM did not affect macrophage activation. Thus, it is believed that low concentrations of LAM stimulate cytokine production, at least initially. However, higher concentrations of LAM (50-100 xcexcg/ml or more) appear to block rather than promote macrophage function. Thus, the production of either too much or too little cytokine at different stages of Mycobacterial disease may contribute to Mycobacterial pathogenesis. New methods for blocking the above-characterized physiological effects of LAM molecules are a highly desirable goal in the treatment of subjects that are or that have been infected with Mycobacteria. For the same reasons, new methods by which fluids containing LAM can be decontaminated prior to administration into a subject are also desirable. Neutralization of even small amounts of LAM is desirable, because small amounts of LAM may have the physiological effect of stimulating cytokine production.
Of interest to the background of the invention are the disclosures of PCT/US88/00510, (WO 88/06038) published Aug. 25, 1988, indicating that certain poloxypropylene/polyoxyethylene nonionic surface-active block copolymers can be used with or without conventional antibiotics to treat infection with Mycobacterium. This reference cites studies suggesting that the effects of other nonionic surfactants on tuberculosis are most likely due to modification of surface lipids of Mycobacteria, and not to direct bactericidal effects on Mycobacteria. See e.g. Cornforth et al., Nature, 168:150-153 (1951).
Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear neutrophils (PMN), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMN""s by acid extraction combined with either ion exchange chromatography Elsbach, J. Biol. Chem., 254:11000 (1979) or E. coli affinity chromatography, Weiss, et al., Blood, 69:652 (1987), and has potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 Daltons (55 kD). The amino acid sequence of the entire human BPI protein, as well as the DNA encoding the protein, have been elucidated in FIG. 1 of Gray, et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference.
BPI has been shown to be a potent bactericidal agent active against a broad range of gram-negative bacterial species. The cytotoxic effect of BPI was originally established to be highly specific to sensitive gram-negative species, with no toxicity being noted for other non-acid fast, gram-positive bacteria or for eukaryotic cells. The precise mechanism by which BPI kills bacteria is as yet unknown, but it is known that BPI must first attach to the surface of susceptible gram-negative bacteria. It is thought that this initial binding of BPI to the bacteria involves electrostatic interactions between the basic BPI protein and negatively charged sites on lipopolysaccharides (LPS). LPS has been referred to as endotoxin because of the potent inflammatory response that it stimulates. LPS induces the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to Lipid A, the most toxic and most biologically active component of LPS.
In susceptible bacteria, it is thought that BPI binding disrupts LPS structure, leads to an activation of bacterial enzymes that degrade phospholipids and peptidoglycans, alters the permeability of the cell""s outer membrane, and ultimately causes cell death by an as yet unknown mechanism. BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its gram-negative bactericidal properties and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, such as bacteremia or sepsis.
An approximately 25 kD proteolytic fragment corresponding to the amino-terminal portion of human BPI holoprotein possesses the antibacterial efficacy of the naturally-derived 55 kD human holoprotein. In contrast to the amino-terminal portion the carboxy-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi, et al., J. Exp. Med., 174:649 (1991). A BPI amino-terminal fragment, expressed from a construct encoding approximately the first 199 amino acid residues of the human BPI holoprotein, has been produced by recombinant means as a 23 kD protein referred to as xe2x80x9crBPI23xe2x80x9d. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
While BPI protein products are effective for treatment of conditions associated with gram-negative bacterial infection, there continues to exist a need in the art for products and methods for treatment of other bacterial infections such as infection with Mycobacteria.
The present invention provides methods of treating a subject suffering from infection with Mycobacteria by administration of a composition comprising a BPI protein product. Therapeutic compositions according to the invention may be administered orally, systemically (such as by intravenous, intramuscular or other injection), or as an aerosol. Mycobacterial disease states subject to treatment according to the invention include tuberculosis, which can be caused by infection with M. tuberculosis, leprosy, which can be caused by infection with M. leprae, and diseases caused by M. avium and other Mycobacteria species. According to preferred methods, anti-Mycobacterial antibiotics such as previously identified and/or surfactants may be administered in combination with the BPI protein product to subjects suffering from infection with Mycobacteria.
According to another aspect of the present invention, compositions comprising a BPI protein product are administered to neutralize LAM""s physiological effects on a host. For example, methods are provided for neutralizing the effect of low concentrations of LAM capable of stimulating cytokine production in a host. Methods are also provided for neutralizing the inhibitory effect that higher concentrations of Mycobacterial LAM (i.e. 100 xcexcg/ml or more) have upon the interferon-mediated activation of macrophages. Specifically, a BPI protein product may be administered to an immunosuppressed subject failing to respond to microbes or tumor cells due to LAM-induced insensitivity of macrophages to activation by T-cell lymphokines.
According to a further aspect of the present invention, a BPI protein product is employed in methods for decontaminating a fluid containing LAM prior to administration of the fluid into a subject. Such decontamination methods of the invention involve contacting the fluid with the BPI protein product prior to administration, under conditions such that LAM forms a complex with the BPI protein product which can be removed from the fluid. Fluids subject to decontamination by the methods of this invention include, but are not limited to, blood, plasma, blood serum, bone marrow, isotonic solutions, pharmaceutical agents, and cell culture agents.
A further aspect of this invention relates to the use of a composition comprising a BPI protein product for the manufacture of a medicament for the therapeutic application of treating any of the aforementioned conditions or infections from which a subject might suffer.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon considering the following detailed description of the invention, which describes presently preferred embodiments thereof.